The hypersensitive response (HR) of higher plants is characterized by the rapid, localized death of plant cells at the site of pathogen invasion. HR occurs during incompatible pathogen/host interactions, such as when a microorganism that normally causes a disease in its host plant infects a non-host plant. The response is associated with resistance against a variety of pathogens, including nematodes, fungi, viruses, and bacteria. For a review of the hypersensitive response, see Dixon et al., Annu Rev Phytopathol 32:479 (1994) and Godiard et al., Curr Opin Genet Dev 4:662 (1994).
The ability of phytopathogenic bacteria to cause HR in resistant or non-host plants is controlled by a cluster of highly conserved bacterial genes named hypersensitive response and pathogenicity (hrp) genes. Most hrp genes are involved in forming a protein secretion apparatus for harpins, heat-stable and proteinaceous proteins which elicit HR when infiltrated into the leaf intercellular spaces of non-host plants. It is known that, when added to a plant cell culture, harpins induce the exchange of H+ and K+ across the plasmalemma to generate active oxygen species (Baker et al., Plant Physiol 102:1341 [1993]).
This invention relates to amphipathic protein-1 (AP-1) whose presence in a plant decreases the extent or duration of HR in a plant. AP-1 can be introduced to or applied to a plant for the purposes of decreasing HR by direct application of isolated AP-1, transient expression of AP-1 by delivery of a nucleic acid or viral vector into a plant cell, or generation of a transgenic plant expressing a foreign AP-1 gene. A foreign gene or nucleic acid sequence is a gene or sequence that has been introduced into a genome by recombinant genetic techniques.
Accordingly, the invention features a transgenic plant whose genomic DNA includes a foreign nucleic acid encoding a polypeptide. In one aspect, a nucleic acid consisting of the foreign nucleic acid hybridizes under stringent conditions to a nucleic acid consisting of SEQ ID NO:1, the coding sequence for an AP-1 (see below) or its complement. In another aspect, the polypeptide includes an amino acid sequence which is at least 70% (e.g., at least 80, 90, or 95%) conserved with or identical to SEQ ID NO:2.
In addition, the expression of the polypeptide in the transgenic plant decreases a hypersensitive response in the transgenic plant, which can be initiated by a pathogenic bacteria in the transgenic plant. An example of a foreign nucleic acid is one that contains the nucleotide sequence of SEQ ID NO:1.
The invention further includes an isolated nucleic acid encoding a polypeptide having an amino acid sequence at least 70% (e.g., at least 80, 90, or 95%) conserved with or identical to SEQ ID NO:2, the amino acid sequence of an AP-1 (see below). In addition, the invention includes an isolated polypeptide having an amino acid sequence at least 70% (e.g., at least 80, 90, or 95%) conserved with or identical to SEQ ID NO:2. The presence of a polypeptide of the invention, or a polypeptide encoded by a nucleic acid of the invention, in a plant decrease a hypersensitive response in the plant.
A xe2x80x9cforeign nucleic acidxe2x80x9d in the context of a genome is any nucleic acid whose sequence is not naturally found in that genome. For example, foreign nucleic acids can be inserted into genomes by means of recombinant DNA technology.
A xe2x80x9cnucleic acidxe2x80x9d encompasses both RNA and DNA, including cDNA, genomic DNA, and synthetic (e.g., chemically synthesized or modified) DNA. The nucleic acid may be double-stranded or single-stranded. Where single stranded, the nucleic acid may be a sense strand or an antisense strand. An xe2x80x9cisolated nucleic acidxe2x80x9d refers to a nucleic acid which may be flanked by non-natural sequences, such as those of a plasmid or virus. Thus, the nucleic acid can include none, some, or all of the 5xe2x80x2 non-coding (e.g., promoter) sequences which are immediately contiguous to the coding sequence. The term, therefore, includes, for example, a recombinant DNA which is incorporated into a vector including an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other sequences. The term also includes a recombinant DNA or RNA which is part of a hybrid gene encoding an additional polypeptide sequence. Moreover, the term is meant to include nucleic acid fragments which are not naturally occurring as fragments and would not be found in the natural state.
By xe2x80x9chybridizes under stringent conditionsxe2x80x9d is meant specific and non-covalent binding to an immobilized reference nucleic acids in the presence of 0.2xc3x97SSC (1.75 g/l NaCl, 0.88 g/l Na3citratexc2x72H2O; pH 7.0) and 0.1% (w/v) sodium dodecylsulfate at 68xc2x0 C.
The term xe2x80x9cisolatedxe2x80x9d as used herein in reference to a given polypeptide means that the polypeptide is substantially free from other compounds, such as those in cellular material, viral material, or culture medium, with which the polypeptide may have been associated (e.g., in the course of production by recombinant DNA techniques or before purification from a natural biological source). Polypeptides are substantially free from other compounds when they are within preparations that are at least 60% by weight (dry weight) the compound of interest. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight the compound of interest. Purity can be measured by any appropriate standard method, for example, by column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.
Where a particular polypeptide or nucleic acid molecule is said to have a specific percent identity or conservation to a reference polypeptide or nucleic acid molecule the percent identity or conservation is determined by the algorithm of Myers and Miller, CABIOS (1989) which is embodied in the ALIGN program (version 2.0), or its equivalent, using a gap length penalty of 12 and a gap penalty of 4 where such parameters are required. All other parameters are set to their default positions. Access to ALIGN is readily available. See, e g., www2.igh.cnrs.fr/bin/align-guess.cgi on the Internet.
The isolation and characterization of an AP-1 gene of the invention will allow the production of variant forms of AP-1 polypeptides which may have advantageous activities such as greater HR-blocking activity or greater protein stability.
Other features or advantages of the present invention will be apparent from the following detailed description, and also from the claims.
The invention relates to amphipathic protein-1, a polypeptide which decreases the extent or duration of HR in plants, e.g., in response to a harpin secreted from a bacterium.
Contemplated within the scope of this invention are recombinant nucleic acids or viruses which allow production of AP-1 in a transformed cell or transgenic organism or allow ease of producing specific or non-specific mutations within the AP-1 reading frame. These recombinant nucleic acids or viruses may further include any one of a variety of sequences upstream of the AP-1 coding sequences, such as strong constitutive promoters; within the AP-1 coding sequence, such as introns containing cis-elements that allow high level expression; or downstream of the AP-1 coding sequence, such as efficient polyadenylation signals. The invention further includes any cells containing or producing such nucleic acids or viruses, and any AP-1 polypeptides produced from such cells.
Also included in the invention are transgenic plants which express or overexpress an AP-1 polypeptide. These plants can be resistant to bacteria-induced HR, as shown in the examples below.